1. Field of the Invention
The present invention relates generally to the fields of nucleic acid transfection. More particularly, it concerns novel polycation:nucleic acid compositions, methods of preparation of such compositions and methods of transfecting cells with such compositions.
2. Description of Related Art
Gene therapy, which involves the delivery and expression of a therapeutic nucleic acid construct to either promote a new gene function, replace a lost gene function, or inhibit current gene function, has evolved into a powerful alternative to many commonly used therapeutics, particularly in the field of cancer therapy (Roth and Cristiano, 1997). The general basis for gene therapy is to transport, deliver, and express a therapeutic nucleic acid construct in a cell to correct abnormal gene function. In cancer gene therapy, while the basis may be the same, a more specific goal often is to mediate cell death in all tumor cells in order to prevent reoccurrence of tumor growth. Unfortunately, the continued development of gene therapy has been slowed not by the ability to identify therapeutic nucleic acid sequences, but by limitations of the delivery composition (Marshall, 1999). The difficulties in obtaining safe and efficient nucleic acid delivery with current nucleci acid vectors has become the single most limiting factor for advancing gene therapy.
The function of the delivery composition is to transport the nucleic acid to the target cell, ensure passage across the target cell membrane, and deliver the nucleic acid into the nucleus for transgene expression. Typically the delivery composition is composed of non-viral or viral components that are used to mediate nucleic acid delivery. In the case of viral components, it is usually replication incompetent or attenuated viruses that are used. Replication defective adenovirus based on serotype 5 is the most commonly used virus in many ongoing cancer gene therapy trials (Rosenberg et al., 2000). This is based on the high transduction efficiency of the delivery composition/vector irrelevant of the cell's replication cycle status. To produce this delivery composition/vector, deletions are typically made in the E1 region of the viral genome (Li et al., 1993). This modification serves two purposes: the delivery composition/vector can then accommodate therapeutic nucleic acid insertion of a limited size, and this renders the virus replication defective.
Unfortunately, the viral genome is still capable of low level expression of viral proteins such as the major hexon coat protein (Yang et al., 1994). This expression occurs at sufficient levels to induce an immune response, which has resulted in continued problems with immunogenicity as well as toxicity (Yang et al., 1994). It has also become apparent that the vector itself is immunogenic and that this immune response may never be overcome in developing future gene therapy delivery compositions based on this virus (Kafri et al., 1998). In addition, the limited size of the inserted nucleic acid has led to problems with utilizing the virus to deliver large or multiple therapeutic nucleic acids. Of greater importance is the fact that this virus is not capable of targeting specific cells, since the expression of the coxackie-adenovirus receptor occurs on many different cell types including both tumor and normal cells (Roelvink et al., 1999). This may even contribute to the virus having a tropism for liver transduction, which can result in hepatotoxicity, particularly if the virus is administered intravenously. Overall, it has become apparent that this delivery composition/vector is immunogenic, difficult to produce economically in large quantities, has a limited therapeutic nucleic acid carrying capacity, a continued dependance upon helper cell lines for production, a lack of targeting, and is still plagued by questions related to safety and toxicity (Marshall, 1999). These issues were recently brought to light when a patient in a clinical trial for ornithine transcarbamylase deficiency died from adenovirus vector related problems (Marshall, 1999).
In contrast to viral delivery compositions, most non-viral delivery compositions are based completely on non-viral components. The three most commonly investigated non-viral delivery composition components are based on formulations involving lipids (e.g., liposomes) (Bendas et al., 1999), polycations (Xu et al., 1998), or simple naked DNA (Chen et al., 2000). Unfortunately, delivery compositions containing these components have had recurrent problems of low transduction efficiency particularly in vivo; naked DNA exhibits the lowest and liposomes exhibit the highest (Bendas et al., 1999; Xu et al., 1998; Chen et al., 2000). In theory, these delivery compositions should also be simple to produce, but there also is a range in ease of delivery composition production; naked DNA requires simple DNA isolation and lipids require complex and extravagant chemical synthesis and formulation (Bendas et al., 1999; Xu et al., 1998; Chen et al., 2000).
Polycations lie in the middle of properties regarding ease of delivery composition production and formulation. Polycations have a self-assembling property when mixed with nucleic acids due to ionic interactions. There have been many studies utilizing the synthetic polycation polyethylenimine (PEI) as a component to deliver nucleic acids to cultured cells as well as to cells in vivo (Bousiff et al., 1995; Boussif et al., 1996; Densmore et al., 2000; Fronsdal et al., 2000; Boletta et al., 1997; Goula et al., 1998; Coll et al., 1999; Kircheis et al., 1997; Hart, 2000; Rudolph et al., 2000). The delivery of plasmids or oligonucleotides has been demonstrated to the brain and kidney (Bousiff et al, 1995; Boletta et al., 1997), and delivery has been demonstrated to the lung (Goula et al., 1998). In addition, this molecule has been used to mediate targeted nucleic acid delivery using proteins such as transferrin that have been chemically coupled to the polycation (Kircheis et al., 1997).
Unfortunately, there are limitations associated with all of these approaches. First, while efficient nucleic acid delivery has been obtained to cultured cells, a survey of the literature shows that a wide range of amine(PEI):phosphate(DNA) ratios are needed to obtain efficient nucleic acid delivery. Second, it has been demonstrated that the direct addition of a targeting ligand to PEI results in targeted, but reduced, nucleic acid delivery (Kircheis et al., 1997). The third and most important limitation is related to toxicity, as high concentrations (amine:phosphate (a:p) ratios of approximately 9:1) of PEI are typically required to obtain high level nucleic acid delivery, but this is at the expense of high level toxicity. When lower amine:phosphate ratios are used, toxicity does drop, but transduction typically drops precipitously when amine:phosphate ratios fall below 6:1.
The utilization of polycations for nucleic acid delivery has led to many different applications for these molecules. One group in particular has been termed, “molecular conjugates” (Cristiano and Roth, 1995). Molecular conjugates are composed of cell and delivery composition specific proteins that have been attached too positively charged polycations. These conjugates bind DNA to form a protein:DNA complex or polyplex (based on the use of polycations) that can target DNA to a specific cell type depending upon the components used. As a result, this delivery composition can consist of at least four types of components that are required for efficient, targeted nucleic acid delivery: a targeting ligand, a polycation for nucleic acid binding:protein attachment, the nucleic acid for RNA and/or protein expression, and an endosomal lysis agent. One drawback of this approach is the large number of components that must be combined to produce an efficient delivery composition. While some components such as the targeting ligand are not absolutely crucial for general nucleic acid delivery, it has been vital for targeted nucleic acid delivery. However, if used, the ligand is attached to DNA using a polycation such as poly-L-lysine (Xu et al., 1998). The ligand and polycation are covalently linked together by one of several different chemicals and is then capable of binding DNA (a polyanion) of any size in a non-damaging, ionic interaction (Xu et al., 1998; Cristiano and Roth, 1995). Unfortunately, problems with low in vivo transduction and high toxicity has limited the use of this vector.
The focus of the work over the past 10 years has been to develop molecular conjugates as efficient, targeted, non-viral delivery compositions for use in gene therapy (Cristiano and Roth, 1995). At least 9 different ligands have been used in molecular conjugates ranging from high molecular weight proteins such as asialoorosomucoid to target hepatocytes, to low molecular weight peptides such epidermal growth factor (EGF) to target lung cancer cells (Xu et al., 1998; Cristiano and Roth, 1995; Cristiano and Roth, 1996). Unfortunately, high level in vitro transduction cannot be obtained with just a targeting ligand, polycation and DNA. It appears to be absolutely crucial that an endosome or vesicle lysis component is incorporated into the delivery composition, especially when receptor-mediated endocytosis or other uptake pathway that results in vesicle formation is utilized for nucleic acid delivery.
Replication defective adenovirus has been used as such an endosome lysis agent.
When targeting with different ligands, high level nucleic acid delivery could only be obtained when the delivery composition was combined with the virus, which is used specifically as an endosome lysis agent, to overcome endosome entrapment of delivery composition (Xu et al., 1998; Cristiano and Roth, 1995; Cristiano and Roth, 1996). Unfortunately, inclusion of the virus causes increased delivery composition related toxicity, size, and immunogenicity, but does not usually increase transduction in vivo (Gao et al., 1993). As a result, the utilization of a molecular conjugate in a delivery composition continues to suffer from three major problems: an inherent degree of complexity based on the number of components required for efficient nucleic acid delivery, the identification of non-viral endosome lysis agents as dependence upon adenovirus for endosome lysis defeats the purpose of developing a non-viral delivery composition, and a lack of efficient in vivo nucleic acid delivery.
Thus, there is still a clear need for nucleic acid delivery compositions that have one or more properties such as low toxicity, high cell transfection efficiency, ease in small or larger scale preparations, and most importantly in cancer gene therapy, the capability of targeting nucleic acid delivery to tumor cells and not normal cells.